Satellite cells are quiescent progenitor cells that are responsible for the regenerative potential of skeletal muscle. In response to injury, these cells activate, proliferate, and subsequently differentiate to form new muscle fibers. Our recent work has demonstrated the importance of the Notch signaling pathway in satellite cell activation. With age, there is a marked decline in the regenerative capacity of muscle. Contributing to this is a diminished response of satellite cells to injury in aged muscle. The primary hypothesis of this proposal is that the age-related impairment of satellite cell activation and the concomitant decline in muscle regenerative potential are due to age-related changes in the Notch signaling pathway. We recently reported that injury to adult muscle leads to a robust upregulation of the Notch ligand, Delta, whereas in aged muscle there is little upregulation of Delta. Forced activation of Notch resulted in enhanced regeneration of aged muscle. Our ongoing studies have also demonstrated that aged satellite cells have an impaired response to Notch signaling compared to adult cells. The focus of this proposal is to further explore age-related changes in satellite cell function and to examine the molecular mechanisms of diminished Notch signaling in aged muscle. In Aim 1, we will examine functional changes of satellite cells with age, including their functional heterogeneity, their propensity to progress along the myogenic lineage pathway, and their ability to self-renew. In Aim 2, the decreased responsiveness of aged satellite cells and their progeny to Notch signaling will be studied in terms of receptor processing, translocation of the active form of Notch from the cytoplasm to the nucleus, and transcriptional responses to activated Notch. In Aim 3, we will examine the expression of Delta in response to injury in adult and aged muscle, testing whether the upregulation of Delta is mediated by the binding of hepatocyte growth factor (which is released from injured muscle) to its receptor, c-met (which is expressed on satellite cells), and the initiation of the Ras/MAPK signaling cascade. We will also characterize regions of the Delta promoter responsible for upregulation of Delta during satellite cell activation using reporter gene assays, gel shift and supershift studies, and site directed mutagenesis. The results of these studies will address not only age-related changes in skeletal muscle but also more global biological questions. Specifically, these studies will explore the role of Notch signaling in tissue morphogenesis postnatally, the biology of tissue-specific stem cells, and mechanisms of impaired tissue maintenance, repair, and regeneration during the aging process.